摘要
目的观察不同浓度二甲双胍对人卵巢颗粒细胞增殖、凋亡和miR-409-3p表达的影响。方法选取2020年6—12月在青岛市妇女儿童医院生殖医学中心进行体外受精/卵胞浆内单精子显微注射(IVF/ICSI)助孕的多囊卵巢综合征(PCOS)病人(PCOS组)和无PCOS不孕病人(对照组)各20例,收集其卵巢颗粒细胞。采用实时荧光定量PCR(Q-PCR)方法检测卵巢颗粒细胞中miR-409-3p的相对表达量。体外培养人卵巢颗粒细胞系KGN,采用CCK8法检测不同浓度(2.5、5.0、10.0、20.0、40.0 mmol/L)二甲双胍作用KGN细胞24、48和72 h的细胞活力,并计算半抑制浓度(IC 50)值;采用流式细胞术检测细胞凋亡率;应用EDU染色试剂盒检测细胞增殖活性;采用Q-PCR方法检测KGN细胞中miR-409-3p表达水平的变化。结果PCOS组的黄体生成素(LH)、LH/卵泡刺激素(FSH)、抗缪勒管激素(AMH)、睾酮(T)和获卵数均显著高于对照组(t=3.49~9.79,P<0.05)。与对照组相比,PCOS组卵巢颗粒细胞中miR-409-3p相对表达量显著降低(t=2.91,P<0.05)。二甲双胍对KGN细胞有抑制增殖和促进凋亡的作用,且具有浓度依赖性;二甲双胍可以促进KGN细胞miR-409-3p的表达(F=556.63,P<0.01)。结论5.0、10.0、20.0 mmol/L的二甲双胍对人卵巢颗粒细胞增殖和凋亡均可发挥调节作用。二甲双胍可能通过上调miR-409-3p的表达调控颗粒细胞的增殖和凋亡,对PCOS排卵异常的恢复可能有积极作用。
Objective To observe the effects of different concentrations of metformin on proliferation,apoptosis,and miR-409-3p expression of human granulosa cells.Methods The granulosa cells were collected from 20 polycystic ovarian syndrome(PCOS)patients(PCOS group)and 20 infertile patients without PCOS(control group)assisted by in vitro fertilization/intracytoplasmic sperm injection(IVF/ICSI)in the Center for Reproductive Medicine of Qingdao Women and Children’s Hospital from Jun to Dec.2020.The expression of miR-409-3p in granulosa cells was detected by Quantitative real time polymerase chain reaction(Q-PCR).Human ovarian granulosa cell line KGN was cultured in vitro.CCK8 was used to detect the cell viability of KGN cells treated with different concentrations of metformin(2.5,5.0,10.0,20.0,40.0 mmol/L)for 24,48 and 72 h respectively,and calculated the semi-inhibitory concentration(IC 50).The apoptosis rate was detected by flow cytometry,cell proliferation activity was observed by EDU staining kit and Q-PCR was used to detect the miR-409-3p expression of KGN cells.Results The luteinizing hormone(LH),LH/follicle-stimulating hormone(FSH),Anti-Mullerian hormone(AMH),testosterone(T)and the number of oocyte obtained in PCOS roup were significantly higher than those in control group(t=3.49-9.79,P<0.05).Compared with the control group,the expression of miR-409-3p in granulosa cells of PCOS group was significantly decreased(t=2.91,P<0.05).Metformin could inhibit the proliferation and promote the apoptosis of KGN cells in a dose-dependent manner.Metformin could promote the expression of miR-409-3p in KGN(F=556.63,P<0.01).Conclusion Metformin of 5.0,10.0 and 20.0 mmol/L can regulate the proliferation and apoptosis of human granulosa cells.Metformin may regulate the proliferation and apoptosis of granulosa cells by up-regulating the expression of miR-409-3p.It suggests that metformin has a positive effect on the recovery of abnormal ovulation in PCOS.
作者
马静
杜鑫
张慧敏
郝翠芳
MA Jing;DU Xin;ZHANG Huimin;HAO Cuifang(Centre for Reproductive Medicine of Qingdao Woman and Children’s Hospital,Qingdao University,Qingdao 266011,China)
出处
《青岛大学学报（医学版）》
CAS
2023年第3期383-388,共6页
Journal of Qingdao University(Medical Sciences)
基金
国家自然科学基金面上项目(81671416)
青岛市医疗卫生重点学科建设项目。
